Phospholemman (PLM), a 72 amino acid single span transmembrane protein, is the major phosphorylation substrate in cardiac membranes in response to adrenergic stimulation. PLM belongs to the newly identified FXYD family of small ion transport regulators. The function of PLM in the heart is unknown except that its mRNA expression is increased after myocardial infarction (Ml). We were the first to show that PLM overexpression affected rat cardiac myocyte contractility and Ca homeostasis; that PLM colocalized with Na/Ca exchanger (NCX1) in cardiac myocytes, that PLM co-immunoprecipitated with NCX1, that PLM overexpression inhibited reverse NCX1 current, and that PLM downregulation enhanced both forward and reverse NCX1 currents. Our HYPOTHESES are: (i) PLM regulates NCX1 and/or Na,K-ATPase (NKA) activities; (ii) phosphorylation is the major mechanism by which PLM regulates NCX1; and (iii) contractile abnormalities post-Mi is partly accounted for by PLM overexpression. Our SPECIFIC AIMS are: (i) overexpress and downregulate PLM in cardiac myocytes and evaluate the effects on NKA activity; (ii) establish a heterologous expression system to study interaction of PLM (and its mutants) and NCX1, by measuring its effects of NCX1 current and Na-dependent Ca uptake, and its ability to co-immunoprecipitate NCX1; (iii) using the heterologous expression system and mutagenesis, establish the region of PLM that is critical for regulation of NCX1; (iv) evaluate the role of PLM phosphorylation on NCX1 inhibition: serine mutants will be expressed in PLM knockout myocytes; (v) evaulate if PLM protein is overexpressed post-Mi and if contractile abnormalities post-Mi are ameliorated by PLM downregulation. We will use 3 model systems: cultured adult rat cardiac myocytes, HEK293 cells, and PLM knockout mouse.We will employ biophysical, biochemical, and standard molecular biological methods. Our proposed studies will generate knowledge regarding novel pathways involved in physiological modulation of NCX1 and NKA, and the role of PLM in pathophysiological states.